Improved electron microscopes, more stable cryo-stages and direct electron detectors with high quantum efficiency have recently brought about a revolution in biological electron microscopy (EM), enabling near atomic resolution 3D structures for large molecules and viruses1-3. Electron cryo-microscopy (cryo-EM) is used to determine protein structures. It is a method still under development and the resolutions of resulting 3D reconstructions have been steadily increasing. With current methodology, near-atomic resolutions can be obtained. As the resolutions increase further, the pharmaceutical industry will hope to exploit this technique, probably in parallel with x-ray crystallography, to understand how drugs bind to their protein targets. Still, grid design and preparation have remained largely unchanged since they were initially developed for electron cryo-microscopy (cryo-EM) almost three decades ago4-6.
There are several problems with the current grids used in cryo-EM of biological specimens. First, the accumulation of electron-deflecting surface charges and beam-induced motion of samples lead to sub-optimal images7-10. Second, the large surface area to volume ratio of the thin water films present just prior to vitrification cause proteins to accumulate at the air-water interface, resulting in denaturation and strongly preferred orientations5,11. The advent of graphene and more recently graphane12,13 allow these long standing problems to be addressed.
Currently, amorphous carbon is often used as a support for electron microscopy. Typically, thin carbon films (20-200 Å thick) are suspended over thicker layers of holey amorphous carbon which span the gaps in a typical metal mesh grid14. These thin carbon films are thought to decrease charging, increase particle concentration due to protein adsorption to the surface, and alter the orientational distributions of molecules.
Glow-discharging, a method using a poorly controlled plasma made by ionizing the residual air gases present after pumping a grid to mTorr pressures, makes the carbon support layer hydrophilic. This gives it adsorptive properties to help create a uniform distribution of proteins in a thin layer of ice for subsequent imaging by cryo-EM4.
Amorphous carbon substrates have several disadvantages. First, they contribute significantly to noise in the images, especially detrimental for smaller molecules (less than 500 kDa) where the signal to noise ratio (SNR) is critical for accurate particle alignment. Second, proteins often adopt orientational preferences due to interactions with the supporting carbon, and these orientations are poorly controlled using current methods. Third, thin amorphous carbon is a poor conductor that accumulates significant mobile surface charge that can deflect the electron beam and exert strong electrostatic forces on the sample. Finally, it is difficult to control the adsorption of proteins onto the surface because of difficulties in reproducing glow discharge conditions, and because contamination builds up on the carbon surfaces dependent on their age and storage environment.
Graphene is a remarkable material, composed of a sheet of carbon atoms only one atom thick, bound together in a hexagonal lattice structure. The bonded structure is the same as in graphite, comprising a network of sp2 bound carbons sharing delocalized electrons. As a result, graphene has remarkable conductive properties, and mechanical strength, making graphene an excellent support film for electron microscopy. Graphene is a mechanically robust conductor that is transparent to electrons at all spatial frequencies up to 1/2.1 Å, and is therefore a possible substrate for imaging nanoscale specimens in the electron microscope. Graphene, which may be suspended graphene, conducts charge ballistically over the sub-micron distances that span a typical hole used for imaging molecules in ice, so it is likely to significantly reduce the buildup of surface charge during electron beam exposure. In contrast, amorphous carbon is a semiconductor whose properties and conductivity (˜10−3 S/cm for evaporated amorphous Carbon) can vary widely depending on the conditions during deposition, which are not well controlled in the typical thermal evaporators used for their production. This makes amorphous carbon prone to surface charging and strongly susceptible to beam-induced chemical changes in its composition even at very low doses of electron exposure. Graphene's in-plane mechanical strength (Young's modulus ˜1 TPa) further recommends it as a stable and robust substrate.
Despite its significant potential advantages, and ease of large-scale manufacture using chemical vapor deposition (CVD), graphene has not been widely adopted for use with biological materials. There are two main reasons for this: graphene is hydrophobic which largely precludes the deposition of proteins from aqueous solutions, and it is susceptible to surface contamination (formed during growth or adsorbed subsequently during handling and storage). Previous attempts to use graphene as a substrate for imaging biological molecules have been made, but rendering the graphene surface hydrophilic required either the use of harsh solvents that are incompatible with most proteins (DMP-30) or required conversion of the graphene to graphene oxide.
A major problem in cryo-EM is the precise control of the distribution of proteins within a thin layer of vitreous ice. During blotting and vitrification, proteins often segregate to the air/water interface or to carbon support membranes.
Graphene oxide has been used for a similar purpose22-24 but graphene oxide presents problems since oxygen scatters electrons more strongly than carbon or hydrogen and therefore contributes nearly as much background signal as thin amorphous carbon. In addition, graphene oxide is an insulator and contains crystal defects that decreases its mechanical strength compared to pristine graphene, making it less able to neutralize accumulated surface charge and less stable as a support for thin layers of ice.
Graphene is not used with biological molecules because its hydrophobic nature precludes their reliable deposition, and because it is easily contaminated. These drawbacks mean that graphene has not been applied in EM of biological molecules or other applications involving adsorption or other attachment of biological molecules, such as sensors or similar applications.
In the unrelated field of semiconductor physics, it has been shown that exposure to a low-energy hydrogen plasma can convert graphene to graphane, its fully hydrogenated form13,26. Hydrogenated graphene has not been used for EM with biological samples.
The present invention seeks to overcome problem(s) associated with the prior art.